| Abstract|| |
Objective: The present study evaluates the immunoexpression of p16 and Ki-67 in cervical squamous intraepithelial lesion (SIL) and carcinomas and correlates their expression with clinicopathological features and HPV-DNA status. Material and Methods: A total 36 included cases of SIL and squamous cell carcinoma (SCC) were subjected to p16 and Ki-67 immunostaining. p16 staining was evaluated depending on grading, distribution, localization pattern, intensity and IHC score. Ki-67 expression was graded based on percentage of positive cells. Results: Incidence of HSIL and SCC cases was found to be significantly increased with parity > 5. p16 grade III diffuse nucleocytoplasmic immunostaining was observed in 62.5% LSIL, 80% HSIL and 87% SCC cases. Significant association of p16 staining intensity, IHC score and Ki-67 indices was noted with increasing grades of SILs and carcinomas. Conclusion: Our experience indicates that a combination of p16 and Ki-67 immunostaining may be useful to determine the severity of dysplastic change.
Keywords: Cervix, HPV, p16, ki-67, SIL, SCC
|How to cite this article:|
Singh P, Kaushik S, Thakur B, Acharya S, Bhardwaj A, Bahal N. Evaluation of variable p16 immunostaining patterns, Ki-67 indices and HPV status in cervical SILs and squamous cell carcinomas: An institutional experience. Indian J Pathol Microbiol 2023;66:63-9
|How to cite this URL:|
Singh P, Kaushik S, Thakur B, Acharya S, Bhardwaj A, Bahal N. Evaluation of variable p16 immunostaining patterns, Ki-67 indices and HPV status in cervical SILs and squamous cell carcinomas: An institutional experience. Indian J Pathol Microbiol [serial online] 2023 [cited 2023 Mar 20];66:63-9. Available from: https://www.ijpmonline.org/text.asp?2023/66/1/63/367970
| Introduction|| |
Cervical cancer is the fourth most common cancer among women, of which India contributes accounting for about one-fifth of the total world burden. It is well established that most common causative factor is persistent infection with one or more oncogenic types of Human Papilloma Virus (HPV) in both precursor and cancerous lesions.,, Routine screening by cervical Pap smear is subjected to marked inter and intraobserver variability as well as relative low sensitivity on a single sample., Although, histology was thought to be gold standard, however more sensitive, specific and reproducible biomarkers are needed for detection of cervical precursor and neoplastic lesions.
P16-ink4a (p16 inhibitor kinase4a) functions to decelerate the cell cycle and promote cellular aging and may be used as a biomarker particularly for high-risk HPV-associated squamous and glandular neoplasia of the lower gynecological tract., Ki-67 (MIB1) protein is present during all the active phases of the cell cycle, determines the growth fraction of a given cell population as well as the degree of SILs and carcinomas.
The present study evaluates the immunoexpression of p16 and Ki-67 in histopathologically diagnosed cases of cervical SILs and carcinomas in small biopsies and hysterectomy specimens correlates the expression of these proteins with clinicopathological features as well as HPV-DNA status, wherever available.
| Material and Methods|| |
The present prospective study was conducted in histopathology section over a period of 22 months from 2018 to 2020. Approval was taken from Institutional Ethical Committee before commencing the study. A total of 31 cases of cervical tissue biopsies and 05 hysterectomy specimens diagnosed as SILs and squamous cell carcinoma (SCC) were included in the study. Specimens reported as inflammatory/metaplastic conditions/mesenchymal lesions/hysterectomy specimens for causes other than SIL or carcinoma/samples with inadequate tissue for immunohistochemistry (IHC) were excluded.
Relevant demographic or clinical details and available HPV-DNA status results were recorded in a predesigned working proforma. The tissue samples received in 10% neutral buffered formalin were grossed as per standard laboratory protocol. Sections of 3-5 microns were prepared from paraffin-embedded blocks using an embedding station (Leica EG1150 H + C) and stained with Hematoxylin and Eosin.
For IHC, additional sections of 2-3 microns were taken on Poly-L-lysine coated slides and subjected to immunostaining for p16INK4 (antibody clone 16P04JC2, BIO-SB, 1:50 dilution) and Ki-67 (antibody clone EP5, BIO-SB, 1:80 dilution) markers. All antibodies were validated and titrated before use. Positive control (urothelial carcinoma tissue) and negative control (without primary antibody) were evaluated along with every batch of 05 included cases.
Immunostaining of p16 was evaluated based on grading (% of positive cells), distribution, localization pattern and staining intensity along with IHC score. A four-class grading method was used to describe the percentage of positively stained cells as follows: <5% cells-0 (Negative); 5-25% cells-Grade I; 26-50% cells-Grade II and >50% cells-Grade III. Staining intensity of p16 was categorized as: Weak (point 1), Moderate (point 2), and Strong (point 3). Positive p16 staining localization pattern was assessed as nuclear, cytoplasmic, and nucleocytoplasmic. Distribution of p16 positive immunoreaction was analyzed as diffuse versus focal pattern.
p16 IHC score was calculated by multiplying the grade of p16 expression (depending upon the % of positive cells) X staining intensity (point given). The range of score was 0-9 and was categorized as 0-3 (low score), 4-6 (intermediate score), and 7-9 (high score). The score of 3 was taken as a cut-off value for defining the low vs high p16 expression. Mean IHC score was calculated in each group of Low-grade squamous intraepithelial lesion (LSIL), High-grade squamous intraepithelial lesion (HSIL) and SCC cases.
Strong nuclear Ki-67 immunoexpression in at least 5% epithelial cells (nuclei of minimum 200 epithelial cells across the whole epithelial layer) in suprabasal location was considered as positive expression. Ki-67 expression was graded as follows: <5% cells-0 (Negative); 5-25% cells-Grade I; 26-50% cells-Grade II and >50% cells-Grade III. Detailed histopathology report with diagnoses and IHC findings of both immunomarkers were recorded in the proforma.
High-risk HPV status of the included patients was evaluated with conventional PCR method using PCR Bench Top Lab System 9600. Data obtained were coded and entered into MS Excel sheet. Results are represented in the form of number, frequency, percentage, and data analysis was done using SPSS V 22.0. Chi-square and Fisher exact tests were used and a statistically significant P value was taken as ≤ 0.05.
| Results|| |
In the present prospective study, out of a total of 36 cases, there were 8 cases (22%) of LSIL, 5 cases (14%) of HSIL and 23 cases (64%) of SCC. Single case of Condyloma accuminatum was considered in LSIL category. The overall age range of patients in this study was between 22-83 years, with a mean of 52.28 years and median of 50.0 years. 80% of cases of HSIL and 65.21% of cases of SCC belonged to >50 years of age group. 91.7% of patients were practicing Hindu religion.
Data related to parity was available for 33 cases and a fair number of patients (41.7%) were in parity 1-3 group; however, incidence of HSIL and SCC cases was found to be significantly increased with parity >5 (P value 0.0119). Abnormal bleeding was the most common (75% cases) presentation followed by visible growth in our study. WHO subtypes of SCC in our study were observed as keratinizing type (60.9%), non-keratinizing type (30.5%), adenosquamous and papillary SCC (4.3% each) [Table 1].
|Table 1: Comparative analysis of immunoexpression of p16 and Ki-67 in the study cases|
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Out of 08 cases of LSILs, p16 grade III diffuse nucleocytoplasmic immunostaining was observed in 05 cases (62.5%), diffuse grade III cytoplasmic staining in 02 cases (25%) and focal only nuclear grade I positivity in single case (12.5%). 80% (4/5) cases of HSIL showed diffuse, grade III, nucleocytoplasmic p16 immunoreaction while 20% revealed only diffuse, grade III, cytoplasmic staining. 87% SCC cases (20/23) exhibited diffuse p16 grade III nucleocytoplasmic positivity while 4.3% (1/23) with diffuse grade III only cytoplasmic reactivity [Figure 1], [Figure 2], [Figure 3], [Figure 4]. 8.7% SCC cases (2/23) were p16 immunonegative (01 case each of keratinizing type and adenosquamous carcinoma) but these cases were not tested for HPV-DNA.
|Figure 1: (a) Photomicrograph showing LSIL [Cervical biopsy, H and E, 100X]. Inset-koilocytosis [HandE, 400X]. (b) Photomicrograph showing p16 moderate diffuse grade III nucleocytoplasmic reaction, LSIL [IHC-p16, 100X]. (c) Photomicrograph showing grade II Ki-67 index, LSIL [IHC-Ki67, 100X]. (d) Photomicrograph showing grade I Ki-67 index, condyloma accumiantum [IHC-Ki67, 100X]|
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|Figure 2: (a) Photomicrograph showing HSIL [Cervical biopsy, H and E, 100X]. Photomicrograph showing p16 strong diffuse grade III nucleocytoplasmic reaction, HSIL [IHC-p16, 100X]. Inset- high power view [IHC-p16, 400X]. (b) Photomicrograph showing p16 strong diffuse grade III nucleocytoplasmic reaction in HSIL and infiltrating tumor cells [IHC-p16, 100X]. (c) Photomicrograph showing grade III Ki-67 index, HSIL [IHC-Ki67, 100X]. (d) Photomicrograph showing grade III Ki-67 index, HSIL [IHC-Ki67,100X]|
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|Figure 3: (a) Photomicrograph showing Keratinizing SCC [Cervical biopsy, H and E,100X]. (b) Photomicrograph showing p16 moderate-strong diffuse grade III nucleocytoplasmic reaction, KSCC [IHC-p16, 100X]. (c) Photomicrograph showing grade III Ki-67 index in KSCC [IHC-Ki67,100X]. (d) Photomicrograph showing scattered p16 positive keratinized tumor cells in necrotic debris [IHC-p16, 400X]. (e) Photomicrograph showing non-keratinizing SCC [Cervical biopsy, H and E, 100X]. (f) Photomicrograph showing p16 strong diffuse grade III nucleocytoplasmic reaction, NK-SCC [IHC-p16, 100X]|
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|Figure 4: (a) Photomicrograph showing papillary SCC [Cervical biopsy, H and E, 100X]. (b) Photomicrograph showing p16 strong diffuse grade III nucleocytoplasmic reaction, papillary SCC [IHC-p16, 100X]. (c) Photomicrograph showing grade III Ki-67 index, papillary SCC [IHC-Ki67, 100X]. (d) Photomicrograph showing adenosquamous carcinoma [Cervical biopsy, H and E, 100X]. (e) Photomicrograph showing negative p16 immunoreaction, adenosquamous carcinoma [IHC-p16, 100X]. (f) Photomicrograph showing grade III Ki-67 index, adenosquamous carcinoma [IHC-Ki67, 100X]|
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75% (6/8) cases of LSIL showed weak to moderate staining intensity and rest 25% cases showed strong intensity. 60% (3/5) HSIL cases showed strong p16 staining, while 40% (1/5) showed moderate intensity. 73.9% (17/23) cases of carcinomas showed strong p16 staining intensity, 13.1% with moderate (01 case of NK-SCC and 02 cases of keratinizing SCC) while only single case of NK-SCC had weak p16 immunostaining. Significant association of staining intensity was noted (P = 0.05) with increasing grades of SIL and carcinoma.
Statistically significant increased p16 expression (IHC score > 3) was noted in high-grade lesions (HSILs and carcinomas) as compared to LSILs (two-tailed P value 0.0067). Diffuse p16 grade III nucleocytoplasmic immunostaining was more frequently noted in HSIL and carcinomas. However, no independent significant association of p16 staining distribution, localization pattern and grading is seen within different groups of cervical lesions [Table 2].
Out of 08 cases of LSIL, 25% showed negative Ki-67 proliferation index, rest 75% cases showed grade I and II Ki-67 indices (03 cases each). Most of the HSIL and SCC cases (80% and 87% respectively) showed grade III Ki-67 proliferation indices [Figure 1], [Figure 2], [Figure 3], [Figure 4]. Ki-67 proliferation fractions were seen significantly increased (P = 0.004) within the various groups and with increasing grade of cervical lesions [Table 2].
Out of 25 patients with available HPV-DNA status, 02 high-risk HPV negative cases (01 case each of keratininzing type and papillary SCC) showed p16 grade III, nucleocytoplasmic, diffuse, strong positivity and grade III Ki-67 indices.
| Discussion|| |
Similar to our results, predominant histologic subtype as keratinizing SCC was noticed by Jain et al. (91%) and Jain et al. (73.5%) in their studies. In contrast, Poste et al. observed large cell non-keratinizing SCC accounting for 65.60% cases in their study. The variable distribution of histological subtypes may be related to the sample type/size, selected patients or geographical distribution.
Much effort has been taken to bring down the cervical cancer mortality through screening programs at various levels of the health services. Although mostly HPV infection is transient but its persistence increases the risk of developing precursor lesions as well as cervical carcinomas. Various studies have evaluated the role of biomarkers including p16 and Ki-67 along with coexisting HPV infections in the diagnosis, progression or prognosis of cervical precursor lesions and carcinomas in the literature searched.
In our experience, only 5.5% of cases revealed negative p16 immunostaining while rest were categorized as p16 immunopositive regardless of the localization of positive staining. Although variable p16 immunoexpression has been described in previous studies depending upon sample size, type of selected patients or diagnoses, classification systems of cervical lesions, histological variants, scoring protocols, technical issues like antibody clone used; however, our findings are broadly in accordance with the results of various analyses in the literature searched.
Gupta et al., described p16 positivity in 50%, 60%, 70%, and 95% cases of CIN I, CIN II, CIN III, and SCCs respectively. However, 20% CIN1 cases showed p16 nuclear and nucleocytoplasmic positivity in contrast to diffuse strong nuclear or nucleocytoplasmic expression in 45%, 55%, and 90% cases of CIN2, CIN3 and SCCs respectively. They also concluded that p16 is significantly up-regulated in the high-grade lesions (CIN2/CIN3 - SCC) while comparing immunoreactivity score with normal cervical epithelium.
Queriroz et al., observed p16 positivity in 9.1% of metaplasia, 66.6% of CIN I, 93.4% of CIN II/CIN III and 100% of carcinoma cases. Studies by Brown et al. and Murphy et al., also showed p16 immunopositivity in 100% cases of CIN I/CIN III/SCC and in 98% cases of CIN II which is almost similar to our findings.
In a study by Kishore et al., p16INK4A positivity (taking cut-off >10% of epithelial cells) was noted in 25% CIN1, 50% CIN2, and 75% cases of CIN3. Almost similar to our study, overexpression of p16 (score > 5) was observed in 25% CIN1 cases, 50% CIN2, 75% CIN3 and 73.33% SCCs (P < 0.05). Yan et al. demonstrated positive p16 immunoexpression in 24.4% of CIN1 and 87.5% of CIN2/3 with a sensitivity of 87.5% and PPV of 76.1%. They found no correlation between p16 expression and HPV status (correlation coefficient = 0.144, P = .190). Klaes et al. showed in their study that 58 of 60 invasive cervical carcinomas expressed p16INK4a positivity (98% SCCs and 86% adenocarcinomas), 5 of which were high-risk HPV negative.
Study by Krishnappa et al. suggested that CIN1 specimens with diffuse p16 immunostaining had higher chances of progression to CIN 2-3 although follow-up of the included patients were not available in our analysis.
Our results concluded a significant progressive rise of Ki-67 proliferation index with the increasing grade of LSIL to SCCs (P value 0.004). Similar to our experience, Gupta et al. in their study of 20 cases of dysplasia and 30 cases of carcinomas, revealed that the mean value of the labeling index increased as the nature of the lesion changed from dysplasia to carcinoma with the difference being extremely statistically significant (P < 0.0001).
Srivastava et al., also described that the expression of Ki-67 increased from normal cervical epithelia to varying severity of CINs to carcinomas. Ki-67 positivity was seen in 93.4% cases of CIN I, 100% cases of CIN II, CIN III, and carcinoma cervix. However, it was statistically significant while comparing between controls and cases but not within the groups. Yan et al. considered Ki-67 positivity when more than 5% of the cells showed strong nuclear staining and found that Ki-67 expression was seen in 35.6% cases of CIN1 and 95.0% cases of CIN2/3 with 95% sensitivity and 70.3% PPV.
According to our experience, negative immunoexpression of p16 and Ki-67 was noticed in 02 cases of carcinoma and 02 LSIL cases respectively. While comparing between grading of immunoexpression of both biomarkers within the groups of cervical lesions in our study, only one case of LSIL showed p16 grade I positivity with negative Ki-67 proliferation index. All HSIL cases showed grade III positivity for p16 as well as Ki-67, except one case with grade I Ki-67 proliferation index [Table 3].
|Table 3: Comparative analysis of immunoexpression of p16 and Ki-67 in the study cases|
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Two cases of carcinoma were p16 immunonegative but with grade III Ki-67 indices, while 3 cases showed p16 grade III positivity with grade II Ki-67 expression. 02 cases of KSCC had diffuse grade III nucleocytoplasmic moderate p16 staining with grade III Ki-67 index. Only one case of NK-SCC had grade II Ki-67 index with diffuse grade III moderate nucleocytoplasmic p16 positivity.
Similar to our observations, Keating et al. showed that overall, a histological diagnosis of SIL correlated strongly (P value < .0001) with both biomarkers (Ki-67 and p16) exhibiting positive scores for Ki-67 and p16 in 68.4% and 100% of LSILs and 94.7% and 100% of HSILs, respectively. Strong diffuse staining for p16 was significantly associated with high-risk HPV-associated lesions. Also in a study by Kanthiya et al. found significantly different expressions of p16 and Ki-67 between CIN2/3 vs CIN1 (P < 0.001 both), and of only Ki-67 between invasive carcinoma vs CIN2/3 (P = 0.008). However, the expression of p16 was not significantly different between invasive carcinoma vs CIN2/3 (P = 0.219).
Hebbar A et al. calculated sensitivity and specificity of p16/INK4a and Ki-67 were 76.2%, 87.5%, 90.5% and 87.5% respectively. The overall agreement of both immunomarkers with histopathological diagnosis was statistically significant (P < 0.05) and the diagnostic accuracy improved when both stains were used in together. Jacob et al. suggested a strong relationship of P16ink4a expression with squamous metaplasia, CIN II, CIN III, and SCCs with a P < 0.05. Also Ki-67 index was significantly associated with SCCs when compared with other groups.
6 cases of LSIL and 3 cases of HSIL were analyzed for HPV status and showed 100% positivity. 02 HPV-DNA negative cases (KSCC and papillary SCC) showed p16 grade III, nucleocytoplasmic, diffuse, strong positivity and grade III Ki-67 indices. 02 p16 immunonegative cases (KSCC and Adenosquamous carcinoma) revealed grade III Ki-67 index but no HPV-DNA evaluation was done for them. Higher rate of HPV positivity may be due to smaller number of cases in our study, although no statistically significant association was noted between HPV-DNA status and various included cervical lesions.
A 4-year surveillance study by Zhang et al. have reported the HPV positivity rates 72.4% for CIN I, 81.4% for CIN II and 88.1% for CIN III. HPV positivity was detected in 82.2% of CIN 1 and 97.5% of CIN 2/3 in an analysis by Yan et al. Benevolo et al. observed significantly increased p16 overexpression in 31%, 90%, and 100% cases of CIN I, CIN II, CIN III/carcinomas respectively with higher percentage (93%) of HPV infection in p16 positive CIN, similar to our study.
Similar to our observations, Nam et al., described stronger p16 and Ki-67 expressions were noted with higher CIN grade. They also found negative p16 expression in 77.7% HPV negative patients (7/9).
In an analysis by Zhong et al., results showed that p16 and Ki-67 positivity ratio had no significant difference in persistent hr-HPV infection, transient hr-HPV infection and hr-HPV uninfection with CIN1 suggesting that both biomarkers are useless in prediction of persistent HPV infection in CIN1. Our study described strong correlation between p16 expression and HPV positivity in cervical precursor lesions as well as SCC; however, the difference was insignificant.
| Limitations|| |
One demerit of the study was that non-neoplastic cervical lesions were not included to compare with precursor/carcinoma cases. All the various patterns of p16 immunreaction except positive cells <5% were categorized as positive expression because of lack of specific validated scoring protocol in the literature searched. Limited number of patients with HPV-DNA status was available because carcinoma patients presented with growth were not preferably tested for HPV.
| Conclusion|| |
In the present study, the immunoexpression level of p16 increased with progressive rise of grade in SILs/carcinomas depending upon the various patterns of p16 immunoreaction. We found statistically significant increased p16 expression (IHC score > 3) in high-grade lesions (HSILs and carcinomas) as compared to LSILs (P value 0.0067). Ki-67 indices were also significantly associated with HSILs and carcinomas and it can be adjunct marker to grade the various lesions especially in histopathological ambiguous cases. Higher HPV positivity rate with consistent p16 positive immunoexpression in our experience indicates that a combination of p16 and Ki-67 may be useful to determine the severity of dysplastic change.
Our results do not allow us to recommend any definite protocols for both immunomarkers in the clinical application or routine surgical pathology practice; however, we suggest further larger elaborated studies to substantiate the role of combined use of p16 and Ki-67 in SILs and cervical cancer evaluation for screening purpose and as clinical prognostic biomarkers.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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Professor, Department of Pathology, SGRRIMandHS, Patel Nagar, Dehradun - 248001, Uttarakhand
Source of Support: None, Conflict of Interest: None
[Figure 1], [Figure 2], [Figure 3], [Figure 4]
[Table 1], [Table 2], [Table 3]